Miniaturization of analytical methods and instrumentation for biomedical and clinical research is an area of burgeoning interest. In many cases, the reduction in size of an analytical procedure or technique often translates to a reduction in analysis time and costs. Miniaturization of analytical methods often paves the way for the use of established technologies in high-throughput applications. Accordingly, microchips have been developed for many applications in order to minimize both the time and space required to perform processes such as drug delivery and clinical diagnostic procedures. Furthermore, the use of microchips is ideal due to the ability to perform rapid separations using small analyte volumes and parallel processing in these devices.
Great efforts have been made to develop fast, cost-effective, high-throughput separation methods for nucleic acid analysis. For example, microchip technology is currently being developed in which rapid thermocycling and electrophoretic separation can be accomplished 10 times quicker than conventional techniques. The microchip platform has the potential for integrating sample pretreatment, target amplification, and detection in a single device. Microfabricated chips have also been developed for electrophoretic separations of nucleic acids and proteins. It has been reported that electrophoretic microchips can be used for separation and sensitive laser-induced fluorescence (LIF) detection, with a variety of clinically-relevant analytes, without loss of diagnostic information relative to procedures conducted with traditional slab-gel electrophoresis.
One advantage of electrophoretic microchips is the ability to move fluids through the chip architecture with nothing more than an applied potential. In addition, directional fluid flow at intersections can be electrokinetically-valved by applying potentials to the correct locations in the microchip. These phenomena are, at least in part, controlled by electroosmotic flow.
Electroosmotic Flow (EOF)
The most commonly used substrate for microchips is glass, due to both its optical properties and the ability to use standard photolithographic techniques to fabricate microstructures. In many ways, glass is an ideal substrate for electrophoretic microchips. It is a good electrical insulator localizing the fields used for electrophoretic separation to the microchannel structures where the separation is to occur. Furthermore, the chemical composition of glass, a mixture of siloxanes and silanol groups, allows for electroosmotic flow (EOF) under the appropriate conditions. EOF is essentially an electric field-driven pumping that generates “bulk flow” as a result of the fact that the silanols on the glass surface can be deprotonated. This produces a negatively-charged wall which, in the presence of a low ionic strength buffer, induces the formation of a double layer of cations (the double electric layer, see FIG. 1); this generates a potential at the surface termed the “zeta (ξ) potential”. Under low ionic strength conditions, in properly sized channels, application of an electric field induces movement of the double layer, generating a bulk-flow within the channel. This phenomenon is described by the formula:μEOF=ξε/η  (eq. 1)where μEOF is the mobility of the bulk flow, ξ is the zeta-potential on the surface of the capillary or microchannel wall, ε is the dielectric constant of the medium and η the viscosity of the medium.
The pKa's of the silanol groups present on the surface of glass substrates are typically between 3 and 8, depending upon the makeup of the glass. This means that the surface charge and, thus, the zeta potential, are affected by the pH of the solution in contact with the wall. Above pH 9, all silanol groups are deprotonated, but in lower pH solutions the surface charge decreases as the pH decreases. The greater the extent of deprotonation of the silanol groups, the higher the ξ-potential and the greater the magnitude of the EOF. In contrast, at low pH (e.g., pH 2), the silanols are completely protonated, the negative charge on the surface is negligible, the ξ-potential is very small and the EOF approaches zero.
The magnitude of the EOF is not only a function of the pH, it is also dependent on the ionic strength of the solution contacting the surface. The ionic strength impacts the thickness of this diffuse layer extending into the lumen of the capillary/microchannel, with the thickness being inversely proportional to the square root of the ionic strength of the solution. Hence, as the ionic strength decreases, the diffuse layer thickness increases and so does the magnitude of the EOF. An approximate thickness for the diffuse layer can be as small as 5-10 Å for an electrolyte concentration of 100 mM, up to a distance of 50-100 nm for an electrolyte concentration of 1 mM. Consequently, EOF magnitude is typically inversely proportional to the buffer concentration.
While glass has many advantages as a substrate for microfluidic devices, there are also drawbacks to the use of glass. The premier disadvantages include: 1) limited control of EOF, 2) thermal properties, 3) surface passivation needed for many applications, and 4) high temperature bonding. The EOF problem arises, as it is often necessary to control the magnitude of EOF to perform successful separations. For example, DNA separations require an EOF as close to zero as possible, where as the separation of carbohydrates requires an adequate EOF. Unfortunately, the main parameters for controlling EOF (pH and ionic strength) cannot always be “dialed in” to generate the requisite EOF because they may not be compatible with the conditions needed for separation of the analytes or for analyte stability. Consequently, there has been an on-going search for controlling EOF with means other than pH and ionic strength.
The thermal properties of glass make it ideal for electrophoretic separation, where Joule heating of the solution, due to the current in the capillary or channel, must be minimized. These same properties become problematic however for some of the desired functionalities that can be integrated into microchips. For example, thermal cycling for polymerase chain reaction (PCR) amplification of DNA is now carried out on microchips. The ability to remove heat from the solution, and the inability to rapidly cool the glass microchip prohibits the ultrafast heating and cooling rates that can be achieved on plastic substrates. This leads to longer cycling times and extends the time required to complete the PCR. In addition, passivation of the glass surface is also a problem with a number of biologically-active molecules. Proteins, in particular, have a proclivity for binding to the charged surface of glass as well as other substrate surfaces. This is problematic with protein separation, as well as with the microchip PCR amplifications. PCR is an enzymatic process driven by Thermus aquaticusis (Taq) polymerse, which can be adsorbed to the surface and inhibit its function.
Fabrication of glass microdevices also presents some problems. While clear-cut protocols exist for wet etching of microstructures into glass, bonding of a glass coverplate plate to an etched glass substrate is less straight-forward. Glass microchips are typically bonded using a procedure that includes heating the glass to temperatures as high as 695° C. to fuse the two glass surfaces. The silanols in the channel become dehydrated at these high temperatures, and the surface characteristics have changed enough that EOF is often not seen unless the surface is extensively treated (strong acid or base) to restore the siloxane groups. Even then, coating of the surface with an ionic polymer is sometimes used to ensure consistent reproducible EOF in microfluidic channels. The thermal bonding process is time consuming, requiring an annealing process in excess of eight hours, and often requires repeated bonding cycles. Because different glasses have different expansion properties, high temperature thermal bonding requires the etched plate and coverplate be prepared from the same glass. This is not always optimal for devices, as different etching properties, optical properties, or available thickness may be desired for the two substrate plates to be bonded. Moreover, the extreme temperatures required for bonding preclude the incorporation of additional features during the manufacturing process. These would include metals, which could be used for electrodes integrated directly into the devices, or any coatings or polymeric structures, which might be desired for passivating or “tuning” the surface in the etched features of the microfluidic devices.
Low temperature bonding processes have been proposed for glass devices, but those procedures require perfectly flat glass, and scrupulous cleaning of the surfaces to be bonded. A second method utilizes sodium silicate materials spin-coated onto the etched surface of the glass wafer. The coverplateplate is then put in place and the assembly heated to 90° C. to cure the silicate adhesive. This method has not been widely used because of the difficulties in preparing and using the sodium silicate solutions.
Other Microdevice Materials
While glass is the predominant material used for electrophoretic microdevices, plastic materials offer advantages in terms of cost and ease of fabrication. Plastics are relatively inexpensive to manufacture, using techniques such as hot embossing or injection molding. This allows the production of disposable devices, which would be beneficial for many applications. There are a number of plastics from which to choose, and bonding can be carried out at relatively low temperatures. Disadvantages associated with plastic microdevices include the optical properties, with some polymeric materials having intrinsic fluorescence and others not being optically clear in the wavelengths of interest. In particular, many plastics absorb in the UV wavelengths, which are important either for absorbance measurements or excitation of many fluorescent compounds. A more important problem is that the surface of the plastics is not well suited for the production of EOF. The surface within the channel must then be treated to provide a surface amenable to formation of the double layer needed for EOF generation.